aggregation of purified recombinant protein on dialysis-what do to? - (May/21/2006 )
Hi friends, I am new to protein expression and purification - i have expressed and purified a recomb. protein of 30kDa under denaturing conditions(8m urea , 20mM NaPo4, 10mM Tris, 1M Nacl, 15% Glycerol , 0.5% triton x100 and 20mM imidazole) and eluted the protein using 150mM imidazole. When i dialyse the protein against 10mM Tris with 0.1% triton x100 or 10mM tris with 200mM NaCl the protein gets aggregated and settles down. when i mix with pippet a cloudy solution is formed but after some time it settles down again. what can be done to prevent this. i have to use this for ELISA and other tests. can i use this as such. Pl help me.
-jeevee-
QUOTE (jeevee @ May 21 2006, 03:10 AM)
Hi friends, I am new to protein expression and purification - i have expressed and purified a recomb. protein of 30kDa under denaturing conditions(8m urea , 20mM NaPo4, 10mM Tris, 1M Nacl, 15% Glycerol , 0.5% triton x100 and 20mM imidazole) and eluted the protein using 150mM imidazole. When i dialyse the protein against 10mM Tris with 0.1% triton x100 or 10mM tris with 200mM NaCl the protein gets aggregated and settles down. when i mix with pippet a cloudy solution is formed but after some time it settles down again. what can be done to prevent this. i have to use this for ELISA and other tests. can i use this as such. Pl help me.
couldnt you dissolve your protein under milder conditions ? (no urea).....if no you should dialize gradually....for example against 6M urea 2-3 hours, 4M urea 2-3 hours....etc.....hope ive helped
-Kathy-